【作者】 胡源；

【导师】 刘克武；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 从马铃薯中分离纯化出酪氨酸酶（Tyrosinase, EC.1.14.18.1）,并对其理化性质进行了研究。首先用乙酸-乙酸钠缓冲液对马铃薯匀浆抽提,经分段盐析,DEAE-Sepharose.F.F离子交换柱层析,Sephadex G-150分子筛层析等纯化步骤,所获酶液经SDS-PAGE检验为单一蛋白区带,说明达到电泳纯。纯化倍数为50,酶比活力为170.03U/mg蛋白,亚基相对分子质量为42KD。以3,4-二羟基苯丙氨酸（L-DOPA）为底物做酶学性质分析,测得Km为3.70mmol/L,最适温度为30℃,最适pH为7.50。酶在pH5.60—8.00范围和40℃以下比较稳定,当温度从20℃升到40℃,相对酶活仅仅下降了4%,而当温度从50℃上升到70℃,相对酶活从84%迅速下降到5%。 马铃薯酪氨酸酶具有单酚酶和二酚酶活性,用L-酪氨酸、L-3,4二羟基苯丙氨酸、邻苯二酚和氯原酸为底物,测得其单酚酶活性较低,并且在纯化过程中活性丧失很快,而二酚酶活性较高并且较为稳定,其中以邻苯二酚为底物时活性最高。酪氨酸酶是一种金属酶,选用Na⁺、K⁺、Mg²⁺、Ca²⁺、Cu²⁺、Hg²⁺、Zn²⁺、Fe²⁺八种金属离子和EDTA作用于酪氨酸酶,发现Na⁺无显著作用,K⁺、Mg²⁺、Ca²⁺、Cu²⁺、Hg²⁺对酶具有不同程度的抑制作用,其中Hg²⁺的抑制作用最强,而Zn²⁺、Fe²⁺具有不同程度的激活作用。选用CaCl₂和KCl作为抑制剂,以L-DOPA为底物,表明CaCl₂属非竞争性抑制,KCl为竞争性抑制,用Lineweaver-Burk作图法求得其抑更多还原

【Abstract】 Tyrosinase(EC.1 .14.18.1) was isolated and purified from potato and its biochemical properties was studied. Potato tyrosinase was purified to electrophoretic homogeneity by homogenate,salting-out, chromatography with DEAE-Sepharose F.F and Sephadex G-150. The purification multiple was 50 and the specific activity was 170.03U/mg. The molecular weight is 42KD with SDS-PAGE. The enzyme kinetic properties against L-DOPA were studied. The Michaelis constant (Km) was 3.70 mmol/L, the optimum pH value of the enzyme was 7.5 and the optimum temperature was 30℃.The enzyme was stable within pH 5.60-8.00 and below 40℃ . The relative enzyme activity only decreased 4% when temperature increased from 20℃ to 40℃, while rapidly decreased from 84% to 5% when temperature increased from 50℃ to 70℃ .Tyrosinase from potato exhibited monophenols and diphenols with different substrates(tyrosine, catechol, L-DOPA and chlorogenic acid). The activity of monophenols is lower than diphenols and unstable during purification. The enzyme showed high substrate specificity towards catechol and was very sensitive to metal ions, Its activity is strongly enhanced byZn2+、 Fe2+and inhibited by Hg2+、 K+、 Ca2+、 Mg2+, while Na+ have no distinct inhibition. Ca2+、 K+ were selected for determining types of inhibition and the results showed that KCl was a competitive inhibitor with inhibition constant of 10mmol/L, CaCl2 was a noncompetitive inhibitor, with inhibition constant of 11.68 mmol/L.Sulfiting compounds, ascorbic acid and kojic acid exhibited strong inhibition on potato tyrosinase.Theorder of inhibition ability is DTT>β -Mercaptoethanol >L-glutathione>cysteine>ascorbic acid> kojic acid>thiourea. The inhibition mechanism by kojic acid was investigated. The preincubation time and temperature did not significantly affect tyrosinase inhbition by kojic acid Kojic acid can inhibited monooxygenase by increasing the lag period of catalysing for L-Tyrosine. Kojic acid exhibited a competitive inhibition against L-DOPA with inhibition constant of 0.17 mmol/L.Sectrophotometric methods demonstrated that kojic acid was capable of reducing quinones to diphenols to prevent the final melanin forming.更多还原